Separation of enantiomers in capillary electrophoresis with contactless conductivity detection

Contactless conductivity detection is successfully demonstrated for the enantiomeric separation of basic drugs and amino acids in capillary electrophoresis (CE). Derivatization of the compounds or the addition of a visualization agent as for indirect optical detection schemes were not needed. Non-charged chiral selectors were employed, hydroxypropylated cyclodextrin (CD) for the more lipophilic basic drugs and 18-crown-6-tetracarboxylic acid (18C6H4) for the amino acids. Acidic buffer solutions based on lactic or citric acid were used. The detection limits were determined as 0.3 microM for pseudoephedrine as an example of a basic drug and were in the range from 2.5 to 20 microM for the amino acids.     

毛细管电泳法快速测定五倍子中的没食子酸

建立了毛细管电泳高频电导法测定没食子酸的方法.探讨了缓冲溶液、有机溶剂添加剂、分离电压等因素对分离检测的影响.在电泳介质为10.0mmol/L Tris-5.0 mmol/L H3BO3-15.0?H5OH,分离电压22.0 kV的优化条件下,5.5 min内即可实现没食子酸的分析,线性范围为3.00～100 μg/mL,检出限为1.0 μg/mL.成功地检测了五倍子中的没食子酸.     

Analysis of amino acids in cell culture supernatant using capillary electrophoresis with contactless conductivity detection

CE-C⁴D methods for the analysis of amino acids (AAs) are presented. Combining the results from two methods with acetic acid and cyclodextrin-based BGEs, 20 proteinogenic AAs could be analyzed using CE. CE-C⁴D was also, for the first time, applied to analyze free AAs in samples of mammalian cell culture supernatant. After dilution as only sample preparation, combining the results of the two CE methods allowed monitoring the concentration changes of 17 AAs in samples taken during the cultivation of CHO cells.     

甲氧苄啶的毛细管电泳快速检测新方法

建立了毛细管电泳高频电导法测定药物和尿液中的甲氧苄啶。考察了各种条件对分离和检测的影响。以4.0 mmol/L HAc 体积分数10%甲醇(pH4.0)为电泳介质,分离电压20.0 kV,重力虹吸进样。在优化条件下,甲氧苄啶峰形良好,出峰时间小于6 min,线性范围为1.5~120.0μg/mL,检出限0.5μg/mL。该方法样品处理过程简单,可用于药物制剂的质量控制和临床检验。     

Evaluation of the detection of biomolecules in capillary electrophoresis by contactless conductivity measurement

The sensitivity of contactless conductivity detection to amino acids, peptides and proteins in CE was studied for BGE solutions of different pH values. The LOD and analytical characteristics were compared for acidic and basic conditions and better results were in most cases found for buffers of low pH values. Linear dynamic ranges varied between two orders of magnitude for amino acids and peptides and three orders of magnitude for larger proteins. The concentration detection limits were found to be between 1.2 and 7.5 microM for the amino acids tested and for the larger molecules they varied between 2.6 microM for leucine enkephalin and 0.2 microM for HSA when using a buffer at pH 2.1.     

Efficient sample clean‐up and online preconcentration for sensitive determination of melamine in milk samples by capillary electrophoresis with contactless conductivity detection

Based on an efficient sample clean‐up and field‐amplified sample injection online preconcentration technique in capillary electrophoresis with contactless conductivity detection, a new analytical method for the sensitive determination of melamine in milk samples was established. In order to remove the complex matrix interference, which resulted in a serious problem during field‐amplified sample injection, liquid–liquid extraction was utilized. As a result, liquid–liquid extraction provides excellent sample clean‐up efficiency when ethyl acetate was used as organic extraction by adjusting the pH of the sample solution to 9.5. Both inorganic salts and biological macromolecules are effectively removed by liquid–liquid extraction. The sample clean‐up procedure, capillary electrophoresis separation parameters and field‐amplified sample injection conditions are discussed in detail. The capillary electrophoresis separation was achieved within 5 min under the following conditions: an uncoated fused‐silica capillary, 12 mM HAc + 10 mM NaAc (pH = 4.6) as running buffer, separation voltage of +13 kV, electrokinetic injection of +12 kV × 10 s. Preliminary validation of the method performance with spiked melamine provided recoveries >90%, with limits of detection and quantification of 0.015 and 0.050 mg/kg, respectively. The relative standard deviations of intra‐ and inter‐day were below 6%. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine contamination in milk products.     

Separation of phenolic acids by capillary electrophoresis with indirect contactless conductometric detection

A new method for the electrophoretic separation of nine phenolic acids (derivatives of benzoic and cinnamic acids) with contactless conductometric detection is presented. Based on theoretical calculations, in which the mobility of the electrolyte co- and counterions and mobility of analytes are taken into consideration, the electrolyte composition and detection mode was selected. This approach was found to be especially valuable for optimization of the electrolyte composition for the separation of analytes having medium mobility. Indirect conductometric detection mode was superior to the direct mode as predicted theoretically. The best performance was achieved with 150 mM 2-amino-2-methylpropanol electrolyte at pH 11.6. The separation was carried out in a counter-electroosmotic mode and completed in less than 6 min. The LODs achieved were about 2.3-3.3 microM and could be further improved to 0.12-0.17 microM by using a sample stacking procedure. The method compares well to the UV-Vis detection.     

Enantioseparation of tartaric acid by ligand‐exchange capillary electrophoresis using contactless conductivity detection

A method for the determination of tartaric acid enantiomers using CE with contactless conductivity detection has been developed. Cu(II) as a central metal ion together with l ‐hydroxyproline were used as a chiral selector, the BGE was composed of 7 mM CuCl_2, 14 mM trans‐4‐hydroxy‐l ‐proline, and 100 mM ε‐aminocaproic acid; the pH was adjusted to 5 by hydrochloric acid. Separation with a resolution of 1.9 was achieved in 9 min in a polyacrylamide‐coated capillary to suppress the EOF. Various counterions of the BGE were studied, and migration order reversal was achieved when switching from ε‐aminocaproic acid to l ‐histidine. With detection limits of about 20 μM, the method was applied to the analysis of wine and grape samples; only l ‐tartaric acid was found.     

Determination of Proteinogenic Amino Acids in Human Plasma by Capillary Electrophoresis with Contactless Conductivity Detection

Capillary electrophoresis (CE) with contactless conductivity detection (CCD) represents a viable alternative to liquid chromatography in the analysis of amino acids (AA) in human plasma. After optimizing the composition of the background electrolyte (BGE), and introducing simple plasma pretreatment to remove spurious protein components, the CE/CCD methods allows determination of 18 from 20 proteinogenic AAs, three nonproteinogenic AAs and creatinine in 60 minutes. AAs are separated in their cationic forms in BGE containing 1.7 M acetic acid and 0.1% hydroxyethylcellulose, pH 2.2. LODs for individual AAs vary in an acceptable range with minimum of 4.3 μM for Arg and maximum of 42.9 μM for Cys. Mean concentrations and concentrations ranges for AAs in human plasma samples from 9 healthy individuals are found to agree well with those determined by an LC method in other two laboratories.     

Rapid determination of partition coefficients of pharmaceuticals by phase distribution and microchip capillary electrophoresis with contactless conductivity detection

A simple microchip CE method integrated with contactless conductivity detection was developed for the direct determination of partition coefficients of selected pharmaceuticals after phase distribution equilibrium. The equilibrium of distribution between two phases for four pharmaceuticals was performed using a 1‐octanol/water system and 1‐octanol/buffer system. During the concentration determination, several major factors affecting detection were investigated in detail for each pharmaceutical to optimize the detection sensitivity. In the optimal conditions, sufficient electrophoretic separation and sensitive detection for each target analyte can be achieved within 40 s. The two systems showed a pH‐dependent partition behavior. Moreover, the measured values showed excellent agreement with those obtained by the traditional shake‐flask method with HPLC–UV detection and literature reports, respectively. The developed method can be successfully applied to measure partition coefficient values of pharmaceuticals and requires much shorter analytical time compared to traditional methods.     

Formamide as solvent for capillary zone electrophoresis

A comprehensive investigation of a number of aspects when using formamide as background electrolyte solvent in capillary zone electrophoresis was presented. It included (i) the change of the ion mobility with ionic strength, (ii) the influence of the ionic strength on diffusion coefficients, and (iii) on the separation efficiency expressed by the maximum reachable plate numbers (when only longitudinal diffusion contributed to zone broadening), (iv) the effect of the solvent on pKa values (taken from the literature) of neutral and cation acids, (v) the establishment of the a pH scale in formamide by dissolving acids with known pKa values and their salts at defined proportion (thus circumventing the problem of calibrating the pH meter), (vi) the agreement between the experimentally derived and the theoretical dependence of the effective mobility on pH, (vii) the uptake of water of this hygroscopic solvent from the humidity of the environment and its consequence to the ion mobilities, pKa values, and the chemical stability of the solvent (e.g., hydrolysis), and finally (viii) the use of conductivity and indirect UV absorption to enable detection of analytes below the optical cutoff of formamide.     

Monitoring of circulating amino acids in patients with pancreatic cancer and cancer cachexia using capillary electrophoresis and contactless conductivity detection

Branched chain amino acids (BCAAs), alanine and glutamine are determined in human plasma by capillary electrophoresis with contactless conductivity detection (CE/C⁴D). The baseline separation of five amino acids from other plasma components is achieved on the short capillary effective length of 18 cm in 3.2 mol/L acetic acid with addition of 13% v/v methanol as background electrolyte. Migration times range from 2.01 min for valine to 2.84 min for glutamine, and LODs for untreated plasma are in the interval 0.7–0.9 μmol/L. Sample treatment is based on the addition of acetonitrile to only 15 μL of plasma and supernatant is directly subjected to CE/C⁴D. Circulating amino acids are measured in patients with pancreatic cancer and cancer cachexia during oral glucose tolerance test. It is shown that patients with pancreatic cancer and cancer cachexia syndrome exhibit low basal circulating BCAAs and glutamine levels and loss of their insulin-dependent suppression.     

Determination of five priority haloacetic acids by capillary electrophoresis with contactless conductivity detection and solid phase extraction preconcentration

A sensitive capillary electrophoretic separation method with contactless conductivity detection (C4D) for analysis of five priority haloacetic acids (HAA5) is presented. The analytes were baseline separated in an electrolyte composed of 20 mM 2-(N-Morpholino) ethanesulfonic acid (MES), 20 mM L-histidine (HIS), and 30 μM cetyltrimethylammonium bromide (CTAB) at pH 6.0 in less than 4 min. A simplified solid-phase extraction (SPE) preconcentration procedure on highly cross-linked polystyrene-divinylbenzene (PS-DVB) type sorbent was developed and optimized with respect to short preconcentration time. HAA5 from a 25-mL sample aliquot of tap and swimming pool water could be preconcentrated in less than 5 min using an in-house made SPE column with recoveries ranging from 23 to 98%. Combining the SPE preconcentration procedure with capillary electrophoretic analysis, the attained limits of detection were between 6.1 and 12.2 μg/L with total analysis time of less than 10 min.     

毛细管电泳高频电导法测定苦参中的苦参碱和氧化苦参碱

建立了用毛细管电泳高频电导法测定苦参药材中苦参碱和氧化苦参碱的方法。对电泳介质的种类、浓度、pH值以及操作电压和进样时间对分离检测的影响进行了研究。缓冲液为2.0mmol LNa2HPO4 1.0mmol LH3PO4 体积分数为25%乙醇(pH6.0),分离电压为16.0kV时可实现较好的分离与检测。苦参碱和氧化苦参碱的线性范围为:25.0～1.00×103μg mL(相关系数分别为0 987和0.999);RSD(n=6)分别为:2.1%和0.70%;检出限分别为10.0和5.00μg mL;回收率分别为92.7%～99.1%和100%～102%。     

Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detection

Twenty underivatized essential amino acids were separated using capillary zone electrophoresis and consequently detected with contactless conductivity detection (CCD). A simple acidic background electrolyte (BGE) containing 2.3 M acetic acid and 0.1% w/w hydroxyethylcellulose (HEC) allowed the electrophoretic separation and sensitive detection of all 20 essential amino acids in their underivatized cationic form. The addition of HEC to the BGE suppressed both, electroosmotic flow and analyte adsorption on the capillary surface resulting in an excellent migration time reproducibility and a very good analyte peak symmetry. Additionally, the HEC addition significantly reduced the noise and long-term fluctuations of the CCD baseline. The optimized electrophoretic separation method together with the CCD was proved to be a powerful technique for determination of amino acid profiles in various natural samples, like beer, yeast, urine, saliva, and herb extracts.     

Using capillary electrophoresis for the determination of organic acids in Port wine

The simultaneous separation and determination of organic acids in several samples of white and red Port wines was performed by capillary zone electrophoresis using indirect UV detection with 2,6-pyridinedicarboxylic acid as a background electrolyte buffer. Operational parameters like migration time, temperature, voltage and capillary length were optimized. Sixteen samples of red wine and four samples of white wine were used to analyze for tartaric, malic, lactic, succinic and acetic acids using glyoxylic acid as the internal standard. The method is rapid, sensitive and quantitative, and time-consuming sample preparation, such as solid-phase extraction or liquid-liquid extraction procedure, is not required.     

Carbon nanotube-enhanced separation of DNA fragments by a portable capillary electrophoresis system with contactless conductivity detection

It was demonstrated that separation of DNA fragments by a CE-contactless conductivity detection system (CE-CCD) could be enhanced with multiple-wall carbon nanotubes (MWCNs) as buffer additive. For HaeIII digest of PhiX174 DNA, optimized MWCN concentration was obtained when the MWCN was above its threshold concentration, at which MWCN could form a network in the buffer as pseudostationary phase to provide additional interaction sites. In the case of larger DNA, MWCN near or below its threshold concentration was enough to provide great improvement of the resolution, which was shown by the separation of the 2-Log DNA ladder. Furthermore, the buffer containing MWCN could provide a more stable baseline in the CE-CCD system, owing to less fluctuation of its conductivity. Compared with CE-UV, CE-CCD with MWCN could provide lower LODs as well as better resolution.     

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.     

Indirect thermo-optical detection for capillary electrophoresis

Indirect thermo-optical detection for capillary electrophoresis is described first. A 20 mW helium-neon laser (632.8 nm) was used to provide the pumping beam and a 2 mW helium-neon laser (632.8 nm) supplied as the probe beam; Methylene Blue dye was used as a background absorber. The addition of ethanol to the background electrolyte solution can be performed to reduce adsorption of Methylene Blue onto the capillary wall. The detection method was applied to the detection of amino acids separated by capillary electrophoresis. The detection limit for lysine was 5 × 10⁻⁶ mol l⁻¹ (signal-to-noise ratio, 2).     

Continuous on-line derivatization and determination of amino acids by a microfluidic capillary electrophoresis system with a continuous sample introduction interface

An automatic, rapid and continuous on-line derivatization system coupled to microfluidic capillary electrophoresis (CE) for the determination of amino acids using o-phthaldialdehyde/N-acetyl-l-cysteine (OPA/NAC) as the derivative agents has been developed. By on-line derivatization, amino acids were automatically and reproducibly converted to the UV-absorbing derivatives, which were separated by capillary zone electrophoresis (CZE). Optimization of derivatization and separation condition was carried out to achieve both good sensitivity and separation efficiency. The separation could be achieved within 4 min and sample throughput rate can reach up to 16 h⁻¹. The repeatability (defined as relative standard deviation, R.S.D.) was 2.56, 2.85, 3.24 and 3.60% with peak area evaluation and 2.93, 3.12, 4.20 and 4.91% with peak height evaluation for arginine (Arg), phenylalanine (Phe), serine (Ser) and glycine (Gly), respectively. The limits of detection (S/N=3) were 10.46, 13.14, 34.39 and 44.79 μmol/l for Arg, Phe, Ser and Gly, respectively. Major advantages of the proposed method include improved precision and efficient automation of the derivatization by the FI system and the enhanced sampling frequencies by the combined FI-CE system.